Antisense technology has been exploited as a potential therapeutic approach for the treatment of a variety of pathological disorders including AIDS and cancer. This strategy is based on the premise that sequence-specific oligodeoxynucleotides can serve as inhibitors of gene expression by a direct interaction with their appropriate mRNA or DNA targets. To improve the effectiveness of antisense oligodeoxynucleotides as therapeutic reagents, a great deal of effort has been placed on the use of structurally modified oligomers. In particular, phosphorothioate oligodeoxynucleotides have been widely used antisense reagents in which a sulfur atom is substituted for one of the nonbridging oxygen atoms bound to phosphorous. Phosphorothioates offer several advantages to phosphodiester oligodeoxynucleotides, including greater serum stability and nuclease resistance. Moreover, phosphorothioates hybridize well to target mRNA and elicit RNase H activity which cleaves the mRNA of the mRNA-DNA duplex. However, the polyanionic nature of phosphorothioate oligodeoxynucleotides has led, in some cases, to uncertainty regarding the extent to which observed biological effects stem from an antisense mechanism. Recent evidence implicating phosphorothioate oligodeoxynucleotides in nonsequence-specific protein interactions, i.e., aptameric binding, has raised questions regarding their specificity for target mRNA[1].
Oligodeoxynucleotides interact with proteins in a complex manner that is dependent upon charge, length or concentration [2,3,4]. The binding of oligodeoxynucleotides to protein may result in protein-oligomer complexes that may confer undesirable consequences for normal cellular physiology. For example, phosphorothioate oligodeoxynucleotides have been shown to form complexes with the heparin-binding growth factors, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF),vascular endothelial growth factor (VEGF) (but not epidermal growth factor (EGF) which has a poor affinity for heparin) [5]. In the case of bFGF, binding of the phosphorothioate oligomer is augmented when four contiguous guanosine residues are present. Studies with the tyrosine kinase receptor, bcr-abl, have shown that direct interaction of an oligomer with the protein leads to a reduction in the phosphorylation levels of both receptor and of downstream signaling proteins [6].
This application sets forth several in vitro assays performed to show that phosphorothioate oligodeoxynucleotides of defined sequence and length interact with two cell surface expressed protein tyrosine kinase receptors, flk-1 and EGFR. These receptors play an important functional role in normal and pathological cellular events. The results show that oligodeoxynucleotides bound receptors differentially and elicited alterations in cellular tyrosine phosphorylation patterns in the presence and absence of their cognate ligands. Treatment of cultured tumor cells with phosphorothioate oligodeoxynucleotides resulted in gross changes in cellular morphology, whereas the in vivo administration of one nonspecific phosphorothioate oligodeoxynucleotide significantly inhibited growth of GBM-18 cells in mouse xenografts. These results highlight the biological potency and demonstrate the potential therapeutic efficacy of non-sequence specific phosphorothioate oligodeoxynucleotides.